Alpha amylase assay

ABSTRACT

Method of measuring the amount of α-amylase in a liquid sample, including the steps of providing an oligosaccharide substrate for α-amylase, the substrate being characterized in that it contains at least 3 glucose units, its reducing-end glucose unit is bonded, via a bond cleavable by α- or β-glucosidase, to a label which exhibits an optically measurable change upon cleavage of the bond, and its terminal glucose unit is bonded to a blocking substituent which inhibits cleavage by exo-enzymes of the bond between the terminal glucose unit and the adjacent glucose unit; contacting the sample with the oligosaccharide substrate and with a first exo-enzyme capable of cleaving the bond between the reducing-end glucose unit and the label, and measuring the optically measurable change as a measure of α-amylase in the sample.

This is a divisional of co-pending application Ser. No. 634,873 filed onJuly 26, 1984, now U.S. Pat. No. 4,649,108.

BACKGROUND OF THE INVENTION

This invention relates to measuring the enzyme α-amylase in biologicalfluids. The measurement of α-amylase in urine and serum is widelyperformed in the diagnosis of pancreatic disorders. A number of assaysdescribed in the literature employ oligosaccharide α-amylase substrates,which are cleaved into smaller chains by α-amylase (which is anendo-enzyme), in conjunction with an exo-enzyme, e.g., α-glucosidase,β-glucosidase, or glucoamylase.

Driscoll et al. U.S. Pat. No. 4,102,747 describes an assay employingoligosaccharides of chain length 4-10 glucose units, with a chromophore(p-nitrophenol, or "pNP") on the reducing end. The chain is "resistantto cleavage by α-glucosidase", and cleavage by α-amylase produces"smaller fragments which are acted upon by α-glucosidase . . . toliberate p-nitrophenol."

Marshall et al. (1977) Clin Chimica Acta 277 describes an assayemploying "modified amylaceous polysaccharides containing blockages tothe action of glucoamylase. Such blockages to exo-enzyme action areconveniently introduced . . . by limited periodate oxidation . . . or bysubstitution of monosaccharide residues." "In the presence of excessglucoamylase, the amount of glucose released . . . is directlyproportional to the amount of α-amylase present." "Glucose wasdetermined by the glucose oxidase method."

A brochure published by Calbiochem-Behring describes an assay, the"Pantrak® E. K. Amylase" method, similar to that of Driscoll et al.,supra.

A biomedix® catalog describes the DeltaTest® Assay, similar to that ofMarshall et al., supra.

Three patents assigned to E. I. DuPont de Nemours and Company (Burns etal. U.S. Pat. No. 4,145,527; Farnham et al. U.S. Pat. No. 4,147,860; andMenson et al. U.S. Pat. No. 4,233,403) describe assays similar to thatof Driscoll et al., supra.

U.K. Pat. Appln. GB No. 2004646 describes an assay employingmaltoheptaose as the α-amylase substrate.

SUMMARY OF THE INVENTION

In general, the invention features a method of measuring the amount ofα-amylase in a liquid sample, including the steps of providing anoligosaccharide substrate for α-amylase, the substrate beingcharacterized in that it contains at least 3 glucose units, itsreducing-end glucose unit is bonded, via a bond cleavable by α- orβ-glucosidase, to a label which exhibits an optically measurable changeupon cleavage of the bond, and its terminal glucose unit is bonded to ablocking substituent which inhibits cleavage by exo-enzymes of the bondbetween the terminal glucose unit and the adjacent glucose unit;contacting the sample with the oligosaccharide substrate and with afirst exo-enzyme capable of cleaving the bond between the reducing-endglucose unit and the label, and measuring the optically measurablechange as a measure of α-amylase in the sample.

In preferred embodiments, the label is a chromophore, a fluorophore, achemiluminescent substituent, or a bioluminescent substituent; the firstexo-enzyme is α- or β-glucosidase or a mixture thereof; and the sampleis also contacted with a second exo-enzyme, most preferablyglucoamylase, whose action is independent of substrate chain length andwhich is incapable of cleaving the bond between the label and thereducing-end glucose unit. Preferred labels are p-nitrophenol,o-nitrophenol (chromophores), coumarin derivatives such as4-methylumbelliferone (a fluorophore), and luciferin (a chemiluminescentsubstituent). Preferably, the substrate has eight or fewer glucoseunits, and most preferably has six or seven. Preferred blockingsubstituents are acetals or ketals, e.g., benzylidene. The reagents usedin the assay are preferably provided in the form of a reagent kit, thereagents preferably being mixed together in a single container.

The assay of the invention works according to the general scheme shownin FIG. 3.

In the scheme illustrated in FIG. 3, the blocking group preventsexo-enzymes from breaking down the substrate, so that in the absence ofα-amylase there will be no color change. α-Amylase, an endo-enzyme,cleaves interval α-1,4 bonds in the substrate, producing smallerfragments which can be acted on by the exo-enzyme, which causes theultimate release of the chromophore.

The assay of the invention is most effective when two differentexo-enzymes are used, particularly when the substrate contains more thanfour glucose units. The scheme shown in FIG. 4 illustrates the action ofα-amylase and two exo-enzymes, glucoamylase and α-glucosidase (in thisscheme, there is an α-linkage between the chromophore and thereducing-end glucose unit; there could just as well be a β-linkage, inwhich case β-glucosidase could be used, or α- and β-linkages, in whichcase a mixture of the two enzymes could be used).

In the scheme illustrated in FIG. 4, the α-amylase acts as previouslydescribed. The two exo-enzymes, glucoamylase and α- or β-glucosidase,cooperate as follows. α- or β-glucosidase is relatively inactive duringthe period immediately following the initial cleavage of the substrateby α-amylase, because its activity is chain-length dependent, i.e., itsactivity is inversely proportional to substrate chain length.Glucoamylase, however, acts quickly to break the polysaccharidefragments into single glucose units, its activity being independent ofchain length. As discussed above, neither enzyme can act until α-amylasehas acted, because of the blocking group bonded to the terminal glucoseunit. When the reducing-end glucose, with its chromophore, has beencleaved from the adjacent glucose unit, the role of glucoamylase iseffectively completed, since glucoamylase cleaves only bonds betweenglucose units, and cannot cleave the bond between the reducing-endglucose and the chromophore. It is at this point that α- orβ-glucosidase, which is capable of cleaving such bonds, plays its majorrole, by releasing measurable chromophore.

The assay of the invention produces a linear result, i.e., opticaldensity (OD) measurements directly proportional to α-amylaseconcentration, following a very short lag time, rendering the assayhighly susceptible to automation. Furthermore, the assay likely reducessusceptibility to inhibition by released glucose, because there is lessreleased glucose present, since the portion of the oligosaccharideremaining blocked following the action of α-amylase remains inert to theexo-enzymes. In addition, the components of the assay can be provided inthe form of a kit which has good stability in storage, because theblocking group prevents degradation of the substrate by α-glucosidase,which can otherwise degrade even long-chain substrates over time.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

We first briefly describe the drawings.

DRAWINGS

FIG. 1 is a graph of OD vs. time for five α-amylase standard samples,generated using an assay of the invention.

FIG. 2 is a graph of OD per min. times total assay vol. vs. samplevolume, generated using an assay of the invention.

FIGS. 3 and 4 are diagrammatic representations of the assay schemes ofthe invention.

STRUCTURE AND SYNTHESIS OF SUBSTRATE

The oligosaccharide portion of the substrate can generally be obtainedcommercially, or can be synthesized using standard enzymic techniques,beginning with starch or cyclodextrins.

The label portion, preferably a chromophore such as p-nitrophenol oro-nitrophenol, or a fluorophore such as 4-methylumbelliferone, can beattached to the reducing-end glucose unit using standard techniques,e.g., those described in Driscoll et al. U.S. Pat. No. 4,102,747, herebyincorporated by reference having 4, 5, 6, and 7 glucose units, andoligosaccharide substrates labeled with p-nitrophenol are commerciallyavailable, e.g., from Cal Biochem Corporation.

The blocking group can be any substituent which prevents exo-enzymesfrom breaking down the substrate. The blocking group works by creating aterminal glucose unit no longer capable of fitting the active site ofthe exo-enzyme. Thus, the size and chemical composition of the blockinggroup are not critical; all that is required is that the lock-and-keyenzyme/substrate interaction is prevented. Virtually any substituentbonded to C2, C3, C4, or C6 of the terminal glucose unit will block theaction of exo-enzymes. Because the addition of blocking groups to C6 iseasiest synthetically, C6 blockage (alone or in conjunction withblockage at C4) is preferred.

One class of blocking groups can replace a hydrogen in the hydroxylgroup of C6 of the terminal glucose unit. Suitable such groups include,e.g., carboxylic acid esters (e.g., acetyl or benzoyl); phosphateesters; sulfonate esters (e.g., toluenesulfonyl or methanesulfonyl);ethers (e.g., benzyl, silyl, and triphenylmethyl); and monosaccharidesother than α-1,4 linked glucose.

Alternatively, the blocking group can be an acetal or ketal blockinggroup, i.e., a group which blocks the C4 and C6 hydroxyls of theterminal glucose unit: ##STR1## where (a) R₁ is H and R₂ is lower (5 orfewer carbon atoms) alkyl; lower (10 or fewer carbon atoms) aryl oraralkyl; or (b) R₁ is lower aryl or aralkyl or lower alkyl, and and R₂,independently, is lower aryl or aralkyl, lower alkyl, or CO₂. Inaddition, blocking techniques such as those described in Marshall etal., supra, can be used.

Synthesis of a blocked substrate for use in the assay of the inventioncan be carried out according to either of the following general schemes;the illustrated chromophore is p-nitrophenol, the blocking group isbenzylidene, and there are n+1 glucose units, where 2≦n≦7: ##STR2##

A substrate containing six glucose units, blocked with benzylidene andlabeled with p-nitrophenol, has the formulaO-(4,6-O-benzylidene-α-D-glucopyranosyl)-(1-4)-O-(α-D-glucopyranosyl)-(1-4)-O-(α-glucopyranosyl)-(1-4)-O-(α-D-glucopyranosyl)-(1-4)-O-(α-glucopyranosyl)-(1-4)-O-(4-nitrophenyl-O-α-D-glucopyranoside),and the structure: ##STR3## where n=4.

Compound (1) was synthesized using general method I, as follows. To 2 mlof freshly dried and distilled pyridine was added 39 mg (0.035 mmol) ofα-(4-nitrophenyl)-maltohexaoside. The temperature was raised to 40° C.and stirring was continued until all material had dissolved (about 15min.). The reaction vessel was degassed with argon (3 evacuation-purgecycles), and then a portion of dried and distilled benzal bromide (4 μL,0.67 equiv.) was added. The temperature was gradually (over 30 min.)increased to 115° C. After 3 h at 115° C. an additional 4 μL (0.67equiv.) of benzal bromide was added. A final 4 μL (0.67 equiv.; 2.0equiv. total) of benzal bromide was added 2.5 h later. The mixture washeated at 115° C. for an additional 2 h, and then was cooled in a 0° C.ice bath. After excess acetic anhydride (1 mL) was added, the reactionmixture was allowed to warm to room temperature and stirred until theacylation was complete as judged by analytical TLC (typically 24-48 h).The solution was then diluted with 15 mL of saturated aqueous NaHCO₃.The aqueous phase was extracted with 10 ml portions of CHCl₃ (5x). Thecombined organic layers were washed with saturated aqueous NaHCO₃ (10ml) and filtered through adsorbent cotton. After evaporative removal ofthe CHCl₃ the remaining pyridine was removed by azeotropic evaporationwith several portions of EtOH. A final trituration with EtOH (2 ml)removed any remaining pyridine and left a crude yellow solid. The crudeproduct was purified by preparative thin layer chromatography on two500μ silica gel plates (96:4 benzene-methanol eluant) to give 24.2 mg(36%) of chromatograpically homogeneous material (R_(f) 0.18).Peracylated α-(4-nitrophenyl)maltohexaoside (that is, acylated startingmaterial) was also present (R_(f) 0.13) but was not recovered.Recrystallization from anhydrous EtOH yielded 21.2 mg of analyticallypure compound (2).

The product of this reaction had the formulaO-(2,3-Di-O-acetyl-4,6-O-benzylidene-α-D-glucopyranosyl)-(1-4)-O-(2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-(1-4)-O-(2,3,6-tri-O-acetyl-.alpha.-D-glucopyranosyl)-(1,4)-O-(2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-(1-4)-O-(2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-(1-4)-O-(2,3,6-tri-O-acetyl-4-nitrophenyl-O-α-D-glucopyranoside),and the structure ##STR4## where n=4, and R=Ac.

Compound (1) was prepared from compound (2) as follows. A solution ofcompound (2) in 2.0 mL of CH₃ OH was treated with 2.0 mL of a saturatedNH₃ in CH₃ OH solution. The resultant mixture was stirred at ambienttemperature for 21 h. After evaporative removal of the NH₃ /CH₃ OH andone trituration with ether 12.2 mg (100%) of solid compound (1) wasobtained. This material was not purified further. Enzymic analysissuggested that this sample contained about 8% of freep-nitrophenylmaltohexoside.

α-AMYLASE ASSAY

A substrate solution was prepared by dissolving 12 mg of compound (1),above, in 12 ml of 50 mM αβ DL glycerol PO₄, pH 7.0 ("Gly PO₄ " buffer).α-Glucosidase in 25% glycerol (1,975 units/ml) was obtained from theDuPont Company and glucoamylase was obtained from the Nova Company,purified by conventional methods, and reconstituted in distilled waterto yield 100 units/ml working solution. The α-amylase used as a standardwas Sigma Enzyme Control 2E.

Individual 1 cm path length cuvettes were made up containing 0.9 mlsubstrate solution, 0.0127 ml α-glucosidase stock solution, and 0.10 mlglucoamylase stock solution. The cuvettes were preincubated at 37° C. ina continuous recording spectrophotometer, and the α-amylase standard wasthen added to each cuvette and ΔOD monitored at 405 nM. The results,from the linear portion (after about 21/2 min), are summarized in thefollowing table:

    ______________________________________                                        Volume of                                                                     Sigma E 2 Δ O.D.         Δ O.D.                                   Standard  per         Total    per min. ×                               Added:    minute:     Volume:  Volume:                                        ______________________________________                                        10 μl  0.028       1.023 ml 0.029                                          25 μl  0.066       1.038 ml 0.069                                          50 μl  0.132       1.063 ml 0.140                                          75 μl  0.184       1.088 ml 0.200                                          100 μl 0.231       1.113 ml 0.257                                          ______________________________________                                    

The data from the above table were used to generate the graphs of FIGS.1 and 2. FIG. 1 shows a substantially linear relationship between amountof α-amylase and ΔOD, only 1-2 min. following the initiation of thereaction, at five volumes. The graph of FIG. 2 is corrected for volumeand shows that the linear relationship holds, between enzymeconcentration and ΔOD.

REAGENT KIT

The reagents used in the assay method of the invention are preferablyprovided in the form of a reagent kit which includes the followingcomponents, admixed in a single container e.g., a glass bottle:

Blocked, labeled substrate (preferably containing 6 or 7 glucose units)

Glucoamylase

α- or β-glucosidase

Activator (preferably a source of Cl⁻ ion, e.g., NaCl or KCl, at aconcentration of about 50 mM)

Buffer (e.g., PIPES or HEPES, at a concentration of about 50 mM)

An anti-glucose interference agent (preferably hexokinase [˜10units/ml], ATP [˜5 mg/ml], and MgCl₂ [˜10 mM].

Substrate, glucoamylase, and α- and/or β-glucosidase are used in amountscomparable to those used in prior α-amylase assays. The remaining listedingredients are conventional.

To assay a sample of urine or serum for α-amylase, water and sample areadded to the reagent kit, and optical density or fluorescence aremeasured and compared to standards.

Other embodiments are within the following claims.

I claim:
 1. A reagent kit for an α-amylase assay comprising, in one ormore containers,an oligosaccharide substrate for α-amylase, saidsubstrate being characterized in that it contains at least 3 glucoseunits, its reducing-end glucose unit is bonded, via a bond cleavable byα- or β-glucosidase, to a label which exhibits an opticaly measurablechange upon cleavage of said bond, and its non-reducing end glucose unitis bonded to a chemical blocking substituent which inhibits cleavage byexo-enzymes of the bond between said non-reducing end glucose unit andthe adjacent glucose unit, a first serum-free exo-enzyme capable ofcleaving the bond between said reducing-end glucose unit and said label,and a second exo-enzyme capable of cleaving a bond in said substratebetween two said glucose units.
 2. The reagent kit of claim 1, whereinsaid second exo-enzyme is glucoamylase and said first exo-enzyme is α-or β-glucosidase or a mixture thereof.
 3. The reagent kit of claim 2wherein said substrate, said first exo-enzyme, and said secondexo-enzyme are provided admixed together in a single reagent container.